SUBSTRATE-SPECIFICITY OF THE PROTEIN-TYROSINE PHOSPHATASES

The substrate specificity of a recombinant protein tyrosine phosphatas e (PTPase) was probed using synthetic phosphotyrosine-containing pepti des corresponding to several of the autophosphorylation sites in epide rmal growth factor receptor (EGFR). The peptide corresponding to the a utophosphorylation site, EGFR988-998, was chosen for further study due to its favorable kinetic constants. The contribution of individual am ino acid side chains to the binding and catalysis was ascertained util izing a strategy in which each amino acid within the undecapeptide EGF R988-998 (DADEpYLIPQQG) was sequentially substituted by an Ala residue (Ala-scan). The resulting effects due to singular Ala substitution we re assessed by kinetic analysis with two widely divergent homogeneous PTPases. A ''consensus sequence'' for PTPase recognition may be sugges ted from the Ala-scan data as DADEpYAAPA, and the presence of acidic r esidues proximate to the NH2-terminal side of phosphorylation is criti cal for high-affinity binding and catalysis. The K(m) value for EGFR98 8-998 decreased as the pH increased, suggesting that phosphate dianion is favored for substrate binding. The results demonstrate that chemic al features in the primary structure surrounding the dephosphorylation site contribute to PTPase substrate specificity.